Proteome quantification has become an increasingly essential component of modern biology and translational medicine. Whether targeted or global, stable isotope incorporation with mass spectrometry (MS) analysis is the primary mechanism by which protein abundance measurements are determined. There are numerous approaches to introduce stable isotopes into peptides—SILAC, isobariC tagging (TMT/iTRAQ), iCAT, etc. In most conventional approaches, however, these methods incorporate heavy isotopes to increase mass by at least 1 Da. SILAC, the quantification gold standard, for example, typically utilizes a 4 Da spacing so as to limit the isotopic cluster overlap of the heavy and light peptides. This requirement limits the quantitative capacity of SILAC to triplex. The reason for this is twofold: (1) the mass of the amino acids can only be elevated to ˜+12 Da and (2) mass spectral complexity is increased as multiple isotopic clusters are introduced.
Isobaric tagging addresses the problem of increases in mass spectra complexity by concealing the quantitative information in the MS1 scan, thereby, permitting a higher level of multiplexing than obtained via conventional SILAC methods. Mc Alister et al. recently report methods for expanding the throughput of methods using TMT isobaric reagents from 6-plex to 8 plex, for example, via techniques that resolve the relatively small isotopic shift resulting from substitution of a 15N for a 13C in the isobaric tagging agents. [See, Mc Alister et al., Analytical Chemistry, accepted manuscript, DOI:10.1021/ac301572t]. Despite the advances in the degree of multiplexing accessible using isobaric tagging, these methods have been demonstrated to be susceptible to certain limitations that impact their use in quantitative analysis for applications in proteomics. First, isobaric methods suffer from severe dynamic range compression and loss of quantitative accuracy due to precursor interference with in the MS/MS isolation window. Precursor interference in isobaric methods, for example, has been demonstrated to significantly degrade the quantitative accuracy of the technique. Second, quantitative data can only be obtained for peptides that are selected for further MS2 analysis When replicate analyses are necessary, therefore, this becomes a serious problem as there is high variation in which peptides are selected for MS2 from one run to the next (˜60%). Third, current isobaric tagging methods are only compatible with collisional activation for dissociation, thus limiting the overall versatility of this technique.
From the foregoing it shall be apparent that a need currently exists for mass spectrometry techniques for proteomic analysis. For example, advanced mass spectrometry techniques are needed that are capable of achieving high degrees of multiplexing necessary for high throughput analysis of protein containing samples. In addition, advanced mass spectrometry techniques are needed that are not susceptible to problems of precursor interference that can impact quantitative accuracy and that are compatible with a range of dissociation techniques including electron capture and electron transfer dissociation methods.